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Cryopreservation and post-thaw differentiation of monocytes enabled by macromolecular cryoprotectants which restrict
Natalia Gonzalez-Martinez1,2,3, Ruben M F Tomás4, Akalabya Bissoyi2,3
1Department of Chemistry, University of Warwick Gibbet Hill Road CV4 7AL Coventry UK.
Insights
New cryoprotectants enhance recovery of THP-1 cells, crucial for immunology research. This method improves cell viability and differentiation capacity after freezing, enabling ready-to-use cells directly from storage.
Area of Science:
- Immunology
- Cell Biology
- Cryobiology
Background:
- THP-1 cells are vital for immunology research, differentiating into macrophages and dendritic cells.
- Standard cryopreservation methods yield poor recovery and reduced function in immune cells like THP-1.
- Existing protocols are not suitable for assay-ready THP-1 cells due to extensive post-thaw culture requirements.
Purpose of the Study:
- To develop an effective cryopreservation method for THP-1 cells in an assay-ready format.
- To improve post-thaw recovery rates and maintain cellular function compared to conventional methods.
- To enable direct use of frozen THP-1 cells, reducing research time and resources.
Main Methods:
- Cryopreservation of THP-1 cells using novel macromolecular cryoprotectants (polyampholytes and ice nucleators).
- Comparison of recovery and differentiation capacity with commercial cryoprotectants (DMSO-alone).
- Cryo-Raman microscopy to analyze intracellular ice formation.
Main Results:
- Macromolecular cryoprotectants significantly enhanced THP-1 cell recovery, doubling it compared to DMSO-alone.
- Post-differentiation macrophage phenotype was comparable to non-frozen controls.
- Cryo-Raman microscopy indicated reduced intracellular ice formation with polyampholytes.
Conclusions:
- A novel cryopreservation strategy using polyampholytes and ice nucleators provides enhanced recovery and function for THP-1 cells.
- This method facilitates the routine banking of assay-ready THP-1 cells, accelerating immunological studies.
- The findings support the use of these cryoprotectants for preserving sensitive immune cell lines.
Abstract:
THP-1 is a monocytic cell line which can differentiate into macrophage and dendritic cells, widely used in immunology. Immune cells are particularly sensitive to cryopreservation, leading to low recovery and/or reduced differentiation capacity compared to non-frozen cells. Current cryopreservation protocols are unsuitable to cryopreserve THP-1 cells in 'assay-ready' format, due to the time and resource intensive culturing steps required post-thaw to recover functional cells. We report the cryopreservation of THP-1 cells in vial and multi-well plate format, with significantly enhanced recovery compared to commercial cryoprotectants. This was achieved using macromolecular cryoprotectants (polyampholytes and ice nucleators) which doubled post-thaw recovery relative to DMSO-alone and improved macrophage phenotype post-differentiation comparable to non-frozen controls. Cryo-Raman microscopy demonstrated that the polyampholytes reduced intracellular ice formation compared to DMSO-alone. These results will enable routine banking and 'assay-ready' THP-1 cells direct from the freezer, accelerating immunological research.
